1. Field of the Invention
The present invention relates to methods and compositions for fabrication of high-density nucleic acid arrays for use in biological assays.
2. Background
Hybridization is a hydrogen-bonding interaction between two nucleic acid strands that obey the Watson-Crick complementary rules. All other base pairs are mismatches that destabilize hybrids. Since a single mismatch decreases the melting temperature of a hybrid by up to 10xc2x0 C., conditions can be found in which only perfect hybrids can survive. Many hybridization experiments can be simultaneously carried out on a single solid support on which multiple nucleic acids xe2x80x9cprobesxe2x80x9d have been immobilized by either covalent or non-covalent methods. The tethered xe2x80x9cprobexe2x80x9d is hybridized with target nucleic acids that usually bear a radioactive, fluorescent label, or haptens that could be visualized by chemiluminescent or other detection methods. The resulting hybrids are separated from the unreacted labeled strands by washing the support. Hybrids are recognized by detecting the label bound to the surface of the support.
Oligonucleotide hybridization is widely used to determine the presence in a nucleic acid of a sequence that is complimentary to the oligonucleotide probe. In many cases, this provides a simple, fast, and inexpensive alternative to conventional sequencing methods. Hybridization does not require nucleic acid cloning and purification, carrying out base-specific reactions, or tedious electrophoretic separations. Hybridization of oligonucleotide probes has been successfully used for various purposes, such as analysis of genetic polymorphisms, diagnosis of genetic diseases, cancer diagnostics, detection of viral and microbial pathogens, screening of clones, genome mapping and ordering of fragment libraries.
An oligonucleotide array is comprised of a number of individual oligonucleotide species tethered to the surface of a solid support in a regular pattern, each species in a different area, so that the location of each oligonucleotide is known. An array can contain a chosen collection of oligonucleotides, e.g., probes specific for all known clinically important pathogens or specific for all known clinically important pathogens or specific for all known sequence markers of genetic diseases. Such an array can satisfy the needs of a diagnostic laboratory. Alternatively, an array can contain all possible oligonucleotides of a given length n. Hybridization of a nucleic acid with such a comprehensive array results in a list of all its constituent n-mers, which can be used for unambiguous gene identification (e.g., in forensic studies), for determination of unknown gene variants and mutations (including the sequencing of related genomes once the sequence of one of them is known), for overlapping clones, and for checking sequences determined by conventional methods. Finally, surveying the n-mers by hybridization to a comprehensive array can provide sufficient information to determine the sequence of a totally unknown nucleic acid.
Oligonucleotide arrays can be prepared by synthesizing all the oligonucleotides, in parallel, directly on the support, employing the methods of solid-phase chemical synthesis in combination with site-directing masks as described in U.S. Pat. No. 5,510,270. Four masks with non-overlapping windows and four coupling reactions are required to increase the length of tethered oligonucleotides by one. In each subsequent round of synthesis, a different set of four masks is used, and this determines the unique sequence of the oligonucleotides synthesized in each particular area. Using an efficient photolithographic technique, miniature arrays containing as many as 105 individual oligonucleotides per cm2 of area have been demonstrated.
Another technique for creating oligonucleotide arrays involves precise drop deposition using a piezoelectric pump as described in U.S. Pat. No. 5,474,796. The piezoelectric pump delivers minute volumes of liquid to a substrate surface. The pump design is very similar to the pumps used in ink jet printing. This picopump is capable of delivering 50 micron diameter (65 picoliter) droplets at up to 3000 Hz and can accurately hit a 250 micron target. The pump unit is assembled with five nozzles array heads, one for each of the four nucleotides and a fifth for delivering, activating agent for coupling. The pump unit remains stationary while droplets are fired downward at a moving array plate. When energized, a microdroplet is ejected from the pump and deposited on the array plate at a functionalized binding site. Different oligonucleotides are synthesized at each individual binding site based on the microdrop deposition sequence.
A popular method for creating high-density arrays utilizes pins that are dipped into solutions of the sample fluids and then touched to a surface. The nucleic acid, e.g., DNA, is typically solubilized in an aqueous medium (also sometimes referred to as an xe2x80x9cinkxe2x80x9d) that contains salts, which are used as components of buffers that are compatible with biological macromolecules. 3xc3x97SSC (450 mM sodium chloride and 45 mM sodium citrate) is a standard printing ink. See, e.g., U.S. Pat. No. 5,807,522 (Example 1).
Printing with 3xc3x97SSC is considered useful since the salt particles that are deposited on the arrays after printing enable the verification of the printing process. This verification can be achieved by an imaging method that uses the principle of compound microscopy to photograph printed grids of an HDA, and then electronically determine the presence or absence of the salt spots printed with DNA. By using an oblique white light source, principally the salt deposits are detected by the imaging method (also referred to herein as the 100% Dot Inspection System) and not DNA. Therefore, if one wishes to use such a verification process it is imperative that the ink contains salts.
However, the use of SSC inks can be problematic. The first problem encountered in manufacturing DNA arrays using a 3xc3x97SSC ink is that the rate of evaporation of the aqueous medium is very high compared to the time required to print multiple slides. This is a major obstacle to scaling up the manufacturing process. Additionally, it has been observed by the present inventors that not only is the 3xc3x97SSC ink incapable of printing the required number of slides but variable arrays result due to a rapidly changing concentration of DNA because of the evaporation of aqueous medium.
Therefore, there is a need for an ink composition for printing HDA of nucleic acids that overcome the disadvantages seen in the art.
In an attempt to solve the problem related to evaporation discussed above, we tested an ink composition consisting of 50% DMSO (dimethylsulfoxide) by volume and SSC at a final concentration of 1xc3x97 (150 mM NaCl+15 mM sodium citrate). This composition was considered optimal since DMSO is an organic solvent with high vapor pressure and it is highly miscible with water. Moreover, DMSO is a hygroscopic solvent and is thereby able to compete with the net losses of the solvent due to evaporation of the aqueous component by absorbing moisture from the air. The 1:1 ratio of DMSO and SSC was found to give minimal evaporation of the ink over several print runs and was successfully used to print various HDAs.
To further test the ink, genetic material was suspended in the 1:1 DMSO:SSC (1xc3x97) composition and used to print human and yeast arrays using the contact printing method. Many arrays were printed on CMT-GAPS(trademark) glass slides (Corning) with this ink for over a period of 2 months and the hybridization performance obtained on these slides was acceptable. However, after xcx9c4-5 weeks, these genetic materials failed to give satisfactory hybridization performance. After repeated failed attempts at printing this DNA, the genetic materials were analyzed for the presence of contaminants that could potentially degrade the DNA (for example the DNase enzyme is a common cause of DNA degradation). Gel electrophoreses of the DNA samples showed that a large number of the fragments did not exhibit their expected mobility. Instead the DNA appeared to be retained in the wells of the gel. Such an anomalous behavior is generally associated with the formation of large aggregates of DNA that are not able to travel through the sieving material of the gel matrix. Based on the gel analysis it was clear that the integrity of the DNA molecules had been compromised. Visual inspection of the ink indicated the presence of particulate matter. Infra-red analysis of these particles indicated the presence of sodium citrate and DNA. Further solubility studies on the ink (in the absence of the DNA) indicated that the ink was inherently unstable and was prone to causing the precipitation of the citrate component. The analytical data suggested that the components of the ink resulted in aggregation of the DNA over a period of time.
We have now surprisingly discovered that lowering the final SSC concentration in the ink to about 0.8xc3x97 (120 mM sodium chloride+12 mM sodium citrate) to about 0.1xc3x97 (15 mM sodium chloride+1.5 mM sodium citrate), and having the DMSO concentration between about 30% to about 80% by volume, results in an ink composition that provides superior adhesion, hybridization efficiency and hybridization response from nucleic acid species that are printed on positively charged substrates for nucleic acid binding (e.g., an aminated surface).
We also discovered that unexpectedly the ink of the present invention having a salt concentration significantly lower than the 3xc3x97SSC industry standard enables superior imaging of a highly dense array of printed spots of DNA using a visible light source device. We have further discovered that the ink composition of the present invention satisfies another need in the art in that it enables the long-term storage of the nucleic acid, and thus facilitates the manufacture of HDAs at a high throughput over a long period of time, e.g., over 7 weeks.
We have further unexpectedly discovered that the ink of the present invention having a DMSO concentration greater than 60% by volume results in increased denaturation of the nucleic acid. Even more unexpected is the discovery that this denaturation increases over time. Thus, a lower concentration of DMSO can be used if the time the DNA is suspended in the ink before printing is increased. For example, double stranded DNA in inks containing DMSO at 50% or greater v/v for 21 days showed signs of denaturation. See, FIGS. 6A, 6B, 7A and 7B.
In one embodiment the present invention provides a method for depositing a nucleic acid on a solid support. The method comprises contacting a solid support with a solution of nucleic acid, the solution comprising about 30% to about 80% dimethylsulfoxide (DMSO) by volume, and sodium chloride and sodium citrate salt containing buffer (SSC) at a final concentration of from about 0.1xc3x97 (15 mM sodium chloride+1.5 mM sodium citrate) to about 0.8xc3x97 (120 mM sodium chloride+12 mM sodium citrate). The composition includes a nucleic acid at a concentration ranging from 0.01 mg/ml to 0.50 mg/ml. Preferably, the solution comprises about 40% to about 80% DMSO by volume and SSC at a final concentration from about 0.1xc3x97 to about 0.5xc3x97. More preferably, the solution comprises about 40% to about 60% DMSO by volume and SSC at final concentration from about 0.25xc3x97 to about 0.5xc3x97. Most preferably, the solution comprises about 50% DMSO by volume and SSC at a final concentration of about 0.25xc3x97. The nucleic acid is preferably a double stranded DNA or an oligonucleotide.
In the method any solid support can be used as long as it is capable of retaining the printed nucleic acid. Preferably, the solid support is a two dimensional solid glass surface (for example, a commercially available 3xe2x80x3xc3x971xe2x80x3 microscope glass slides made of soda lime or other glass compositions) or a three dimensional porous glass surface (for example, Vycor(trademark), (Corning Inc.)) or a porous glass substrates made from porous pyrex glass by tape-cast or sol-gel processes. It is preferred that the glass have a surface that is coated to facilitate the adhesion of the nucleic acid. An aminated surface coating is preferred. The aminated surface can comprise, for example, gamma-aminopropyl silane or polylysine.
The contacting step of the method includes, for example, immersing the tip of a pin into the nucleic acid ink solution. The pin can be solid or hollow. The tip of the pin is then removed from the solution to provide solution adhered to the tip. The solution adhering to the tip is then contacted with a solid support to thereby transfer the solution from the tip to the solid support. To form a nucleic acid patterned in an array the contacting step is repeated a plurality of times. This can be accomplished, for example, by use of a typographic pin array.
To facilitate denaturation of the nucleic acid, the nucleic acid may be suspended in the DMSO/SSC composition for at least 1 day prior to printing, preferably at least 5 days, more preferably at least 10 days, still more preferably at least 15 days.
In another embodiment, the present invention provides a nucleic acid printing ink composition comprising about 30% to about 80% dimethylsulfoxide (DMSO) by volume, SSC at a final concentration of from about 0.1xc3x97 to about 0.8xc3x97 and water. The composition can further include a nucleic acid at a concentration ranging from 0.01 mg/ml to 0.5 mg/ml. The nucleic acid is preferably a double stranded DNA or an oligonucleotide. The composition may also include EDTA in a final concentration between 0 and 4 mM, preferably 0.5 mM.
Other agents can be incorporated as part of the ink composition including those that would change the viscosity of the ink for enhancing wettability for certain printing conditions, for example, glycerol, poly-ethylene glycol, histone proteins etc. The inks may also contain small amounts of polycationic agents such as poly-lysine, spermine etc.
Preferably, the solution comprises about 40% to about 80% DMSO by volume and SSC at a final concentration from about 0.1xc3x97 to about 0.5xc3x97. More preferably, the solution comprises about 40% to about 60% DMSO by volume and SSC at final concentration from about 0.25xc3x97 to about 0.5xc3x97. Most preferably, the solution comprises about 50% DMSO by volume and SSC at a final concentration of about 0.25xc3x97. The nucleic acid is preferably a single or double stranded DNA or an oligonucleotide.
Other aspects of the invention are disclosed infra.